Protein Immobilization

We offer a variety of activated supports and accessories for the immobilization of proteins, antibodies, and other molecules. These resins are available separately or in convenient kits. Choose resins based on NHS-ester, AminoLink™, SulfoLink™, GlycoLink™, CarboxyLink™, and other activation technologies.

Thermo Scientific Pierce NHS-Activated Agarose more effectively couples dilute samples than competing products. Rabbit IgG (0.5 mg) was applied to 0.25 mL of activated agarose in PBS at pH 7.2 for 30 minutes. The protein concentration of the flow-through and washes (uncoupled IgG) was determined using the Pierce 660nm Protein Assay. The bound IgG was calculated by subtracting the amount of uncoupled IgG from the amount added. The coupling efficiencies of both dry and slurry forms of Pierce NHS-Activated Agarose were higher than NHS-activated Sepharose™ 4 Fast Flow (GE Healthcare) and Affi-Gel™ 10 Gel (Bio-Rad).

Figure 1. TCEP effectively reduces peptide sulfhydryls (cysteine residues) to maximize covalent immobilization efficiency to SulfoLink Coupling Resin. Peptide A (0.5 mg) and human calcitonin peptide (1 mg) were incubated with (+) or without (-) 25 mM TCEP for 30 minutes and then reacted to 2 mL of SulfoLink Resin for 30 minutes using the default kit procedure. Each peptide contained a C-terminal cysteine, as well as an N-terminal fluorescent tag by which the proportion of non-coupled peptide was determined upon recovery of the reaction solution.

High-yield protein coupling

Immobilization efficiencies for various proteins on 1 mL of AminoLink Plus Resin when immobilized using the pH 7.2 coupling procedure.

Protein

Protein (mg) reacted

Protein (mg) coupled

Coupling efficiency (%)

Protein G

4.6

4.0

83

Mouse IgG

4.7

4.5

96

Rat IgG

4.7

4.4

93

GAM*-IgG

0.9

0.8

84

Human IgG

4.8

4.6

97

Human IgM

0.9

0.8

93

*GAM-IgG is goat anti-mouse IgG, a secondary antibody.

Additional activated supports and accessories are available, as well as convenient kit formats that included disposable columns, buffers, and coupling reagents. Some activated supports are also available in magnetic bead formats.